Anti-Mycoplasma spp. subunit vaccine

ABSTRACT

Provided in the present invention are anti- Mycoplasma  spp. subunit vaccines, especially proteins suitable for being used as the active ingredient of the  Mycoplasma  spp. subunit vaccines, and a vaccine prepared therefrom. Upon experimenting, it is confirmed that the proteins can elicit an immune response having sufficient strength to avoid the infection of  Mycoplasma  spp. in pigs. The vaccine can comprise one of the aforementioned proteins as an active ingredient, or can comprise two or more of the proteins to form a form of cocktail vaccine. The vaccine of the present invention is not only more safe than conventional vaccines, but also has equivalent or even better immune effects.

FIELD OF THE INVENTION

The present disclosure relates to a vaccine against Mycoplasma spp.;especially to a subunit vaccine against Mycoplasma spp.

BACKGROUND OF THE INVENTION

Mycoplasma spp. is currently known the tiniest bacteria capable ofself-replication outside host cells. Although swine enzootic pneumoniawould not cause swine death, it will reduce feeding efficiency and causegrowth retardation, inflammation, and immunosuppression as well as makeswine more vulnerable to infection of other pathogens, which thereforebecome economic damage of the industry.

So far, swine enzootic pneumonia is prevented by three major strategies,including: medicine administration, environment management, andvaccination. Seeing the bad prevention efficiency of antibiotics toMycoplasma hyopneumoniae, medicine administration can only used fortreatment purposes and is hard to meet prevention needs. Furthermore,considering that drug abuse may lead to a larger infection causing bydrug-resistant bacteria, medicine administration needs cautious plansand exists a lot of limitations.

Environment management forms the basis of prevention of Mycoplasma spp.infection. Good piggery sanitation and management would be helpful toreduce occurrence of infection. On the other hand, prevention could bemore comprehensive through vaccination.

The conventional vaccines in the field use inactive/dead bacteria as theactive ingredient thereof. However, the price of the conventionalvaccines is too high because Mycoplasma spp. is fastidious bacteria andis difficult to be cultured in the laboratory. In order to reduce thecost of Mycoplasma spp. vaccines, scientists continuously try to developvaccines of different types, such as: (1) attenuated vaccines, (2)vector vaccines, (3) subunit vaccines, and (4) DNA vaccines. Among them,subunit vaccines show the most potential because the advantages of easein production and high safety.

To date, there are several potential candidate proteins that could beused for M. hyopneumoniae vaccines; however, there is no further reportverifying the proteins suitable for M. hyopneumoniae vaccines.

SUMMARY OF THE INVENTION

In light of the foregoing, one of the objects of the present inventionis to provide antigens suitable for being used in M. hyopneumoniaevaccines and thereby producing novel M. hyopneumoniae vaccines so thatthe cost of prevention can be reduced.

Another object of the present invention is to provide a combination ofantigens that suitable for being used in M. hyopneumoniae vaccines andthereby provide subunit vaccines with better performance; therefore,there would be more options for prevention tasks.

In order to achieve the aforesaid objects, the present inventionprovides a recombination protein for preparing a vaccine for preventingMycoplasma spp. infection, comprising an amino acid sequence of SEQ IDNO: 08, SEQ ID NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQID NO: 13, SEQ ID NO: 14, or a combination thereof.

The present invention also provides a vaccine for preventing Mycoplasmaspp. infection, comprising: an active ingredient, comprising a proteinof PdhA, XylF, EutD, Mhp145, P78, P132, Mhp389, or a combinationthereof; and a pharmaceutically acceptable adjuvant.

Preferably, said active ingredient is of a concentration of 50 to 3500μg/mL based on the total volume of said vaccine.

Preferably, said active ingredient comprises at least two proteinsselected from a group consisting of PdhA, XylF, EutD, Mhp145, P78, P132,and Mhp389.

Preferably, said active ingredient comprises PdhA and P78.

Preferably, said active ingredient comprises XylF and Mhp145.

Preferably, said pharmaceutically acceptable adjuvant is a completeFreund's adjuvant, an incomplete Freund's adjuvant, an alumina gel, asurfactant, a polyanion adjuvant, a peptide, an oil emulsion, or acombination thereof.

Preferably, said vaccine further comprises a pharmaceutically acceptableadditive.

Preferably, said pharmaceutically acceptable additive is a solvent, astabilizer, a diluent, a preservative, an antibacterial agent, anantifungal agent, an isotonic agent, an absorption delaying agent, or acombination thereof.

The present invention further provides a vaccine for preventingMycoplasma spp. infection, comprising: an active ingredient, comprisingan amino acid sequence of SEQ ID NO: 08, SEQ ID NO: 09, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or acombination thereof; and a pharmaceutically acceptable adjuvant.

Preferably, said active ingredient is of a concentration of 50 to 3500μg/mL based on the total volume of said vaccine.

Preferably, said active ingredient comprises at least two amino acidsequences selected from a group consisting of SEQ ID NO: 08, SEQ ID NO:09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQID NO: 14.

Preferably, said active ingredient comprises amino acid sequences of SEQID NO: 08 and SEQ ID NO: 12.

Preferably, said active ingredient comprises amino acid sequences of SEQID NO: 09 and SEQ ID NO: 11.

Preferably, said pharmaceutically acceptable adjuvant is a completeFreund's adjuvant, an incomplete Freund's adjuvant, an alumina gel, asurfactant, a polyanion adjuvant, a peptide, an oil emulsion, or acombination thereof.

Preferably, said vaccine further comprises a pharmaceutically acceptableadditive.

Preferably, said pharmaceutically acceptable additive is a solvent, astabilizer, a diluent, a preservative, an antibacterial agent, anantifungal agent, an isotonic agent, an absorption delaying agent, or acombination thereof.

The present invention more provides an expression vector for preventingMycoplasma spp. infection, comprising: a plasmid; wherein said plasmidcomprises: a nucleotide sequence comprising at least one sequenceselected from a group consisting of SEQ ID NO: 01, SEQ ID NO: 02, SEQ IDNO: 03, SEQ ID NO: 04, SEQ ID NO: 05, SEQ ID NO: 06, and SEQ ID NO: 07;and a regulatory element.

Preferably, said regulatory element comprises a promoter and a ribosomebinding site.

Preferably, said plasmid is pET-MSY, pET-YjgD, pET-D, or pET-SUMO.

Preferably, said plasmid further comprises a gene encoding a fusionpartner.

Preferably, said fusion partner is msyB of E. coli, yjgD of E. coli,protein D of Lambda bacteriophage, or SUMO of S. cerevisiae.

Preferably, said expression vector is used for an E. coli geneexpression system.

To sum up, the present invention is related to antigens that aresuitable for being used as the active ingredient of a M. hyopneumoniaesubunit vaccine and a M. hyopneumoniae subunit vaccine/compositionprepared by using the same. The present subunit vaccine not only can beeffectively used in prevention task for lowering down the cost thereof,the disclosure of the present invention also shows that a “cocktail”subunit vaccine (i.e. having at least two antigens as activeingredients) using at least two antigens of the present invention hasimproved induction of immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one color drawing.Copies of this patent or patent application publication with colordrawing will be provided by the USPTO upon request and payment of thenecessary fee.

FIG. 1 shows the result of the two-dimensional gel proteinelectrophoresis conducted in the 1^(st) example of the presentinvention.

FIG. 2 shows the result of the color reaction of the Western blotconducted in the 1^(st) example of the present invention.

FIG. 3 shows the result of the electrophoresis of the PCR productsobtained in the 2^(nd) example of the present invention.

FIG. 4 shows the records of the challenge experiments conducted in the3^(rd) example of the present invention.

DESCRIPTION OF REFERENCE SIGNS IN THE FIGURES

-   -   1 XylF (xylose-binding lipoprotein)    -   2 XylF (xylose-binding lipoprotein)    -   3 XylF (xylose-binding lipoprotein)    -   4 PdhA (pyruvate dehydrogenase E1-alpha subunit)    -   5 Mhp145 (periplasmic sugar-binding protein)    -   6 EutD (phosphotransacetylase)    -   7 EutD (phosphotransacetylase)    -   8 Mhp389    -   9 P78 (lipoprotein)    -   10 P132

DETAILED DESCRIPTION OF THE INVENTION

One of the core concepts of the present invention is to survey potentialcandidate antigens suitable for subunit vaccines by usingtwo-dimensional gel protein electrophoresis along with immunologicalscreening technology and to identify the antigens by mass spectrometer.Then, the performance of the present subunit vaccines were verified byanimal model experiments.

Briefly, the progress of the development of the present invention is:

(1) Inducing immune response of experiment pigs by injecting aconventional M. hyopneumoniae vaccine and obtaining serum containinganti-M. hyopneumoniae antibodies. (2) Obtaining total proteins of M.hyopneumoniae for two-dimensional gel protein electrophoresis. (3)Conducting hybridization of the result of the two-dimensional gelprotein electrophoresis of step (2) by using the serum of step (1) as1^(st) antibody, and then collecting proteins showing positive (i.e.candidate antigens) from the gel after amplification by a 2^(nd)antibody and the following development procedure. (4) Identifying thecandidate antigens obtained in step (3). (5) Expressing said candidateantigens in large amounts by using an E. coli gene expression system.(6) Examining the efficacy of the present subunit vaccines in reducingpathological traits in lung by swine challenge experiments and therebyverifying the value of said candidate antigens in being used as activeingredient of a subunit vaccine.

The present vaccine for preventing Mycoplasma spp. infection comprisesan active ingredient and a pharmaceutically acceptable adjuvant.

In an embodiment of the present invention, said active ingredient may bePdhA, XylF, EutD, Mhp145, P78, P132, or Mhp389. In an alternativeembodiment, as long as the antigenic determinant of any of the aforesaidprotein is not interfered, said active ingredient may be a fusionprotein of any two of the aforesaid proteins. In another alternativeembodiment, said active ingredient comprises at least two of theaforesaid proteins; that is, so called a “cocktail” vaccine of thepresent invention.

In another embodiment of the present invention, said active ingredientmay comprise an amino acid sequence of SEQ ID NO: 08, SEQ ID NO: 09, SEQID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,or a combination thereof. In an alternative embodiment, as long as theantigenic determinant formed by folding of a peptide of said amino acidsequence is not interfered, said active ingredient may be a fusionprotein with at least two said sequences. In another alternativeembodiment, said active ingredient comprises two or more proteinsrespectively comprising one of the aforesaid amino acid sequences; thatis, so called a “cocktail” vaccine of the present invention.

Said pharmaceutically acceptable adjuvant is used for improving theimmune effect of said active ingredient, stabilizing said activeingredient, and/or increasing the safety of vaccines. Saidpharmaceutically acceptable adjuvant of the present invention includes,but not limits to: a complete Freund's adjuvant, an incomplete Freund'sadjuvant, an alumina gel, a surfactant, a polyanion adjuvant, a peptide,an oil emulsion, or a combination thereof.

The vaccine of the present invention may have one or at least two saidactive ingredients (i.e. a cocktail vaccine). In an example of thepresent vaccine, said active ingredient is of a concentration of 50 to3500 μg/mL based on the total volume of said vaccine. In a preferableembodiment of the present invention, when said vaccine comprises onlyone said active ingredient, said active ingredient is of a concentrationof 50 to 500 μg/mL based on the total volume of said vaccine. In analternative embodiment of the present invention, the present vaccinecomprises at least one said active ingredient; wherein the totalconcentration of said active ingredient(s) contained in said vaccine is50 to 1000 μg/mL, 50 to 1500 μg/mL, 50 to 2000 μg/mL, 50 to 2500 μg/mL,50 to 3000 μg/mL, or 50 to 3500 μg/mL based on the total volume of saidvaccine.

Another aspect of the present invention is to provide an expressionvector for preventing Mycoplasma spp. infection. Specifically, saidexpression vector may be used for an E. coli gene expression system.Nevertheless, without being apart from the spirit of the presentinvention, those having ordinary skill in the art can modify said vectorbased on the disclosure of the present invention and make said vectorsuitable for different gene expression system while still belongs to thescope of the present invention.

Said expression vector comprises a plasmid. Said plasmid comprises: anucleotide sequence comprising at least one sequence selected from agroup consisting of SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ IDNO: 04, SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, and a combinationthereof; and a regulatory element.

Said vector is used in an E. coli gene expression system and forproducing the antigens of the present invention via E. coli. In otherwords, said nucleotide sequence can be translated into the aminosequence of the present antigen via an E. coli gene expression systemand then the amino acid sequence can fold into the present antigen.

In an alternative embodiment, as long as the operation of the E. coligene expression system is not hindered and the production of saidnucleotide sequence and the folding of the consequent amino acidsequence thereof are not interfered, said plasmid may comprise two ormore said nucleotide sequences.

Said regulatory element is referred to an element required forinitiating the transcription and translation in the expression system.Said regulatory element shall at least comprise a promoter, and aribosome binding site. Preferably, said regulatory element may furthercomprise: an operator, an enhancer sequence, or a combination thereof.

In a preferable embodiment of the present invention, said plasmidfurther comprises a gene encoding a fusion partner. Said fusion partnerincludes but not limits to msyB of E. coli, yjgD of E. coli, protein Dof Lambda bacteriophage, or SUMO of S. cerevisiae. Said MsyB is rich inacidic amino acid and might be favorable for improving the solubility ofthe proteins to be produced.

The following examples recite the trials and experiments of the presentinvention in order to further explain the features and advantages of thepresent invention. It shall be noted that the following examples areexemplary and shall not be used for limiting the claim scope of thepresent invention.

Example 1 Screening for Candidate Antigens Suitable for being Used asActive Ingredient of a Subunit Vaccine

Preparation of Serum Containing Anti-Swine Mycoplasm Spp. Antibody.

According to researches, there are seven Mycoplasm spp. can be isolatedfrom swine: Mycoplasm hyopneumoniae, Mycoplasma hyorhinis, Mycoplasmahyosynoviae, Mycoplasma flocculare, Mycoplasma hyopharyngis, Mycoplasmasualvi, Mycoplasma bovigenitalium (Gourlay et al., 1978; Blank et al.,1996; Assuncao et al., 2005). Among them, M. hyopneumoniae is the majorpathogen of swine enzootic pneumonia with an infection rate of 25 to93%. Therefore, the present invention used M. hyopneumoniae (PRIT-5strain) for immune proteomics studies and as sources of genes encodingantigens. Friis medium (Friis et al., 1975) as used for culturing M.hyopneumoniae. According to the experiment design, a proper amount ofantibiotic or agar of 1.5% was added to formulating a solid medium.

Three SPF pigs of 4-week old were brought from Agricultural TechnologyResearch Institute and fed with same feed and kept at same environmentand growth condition in piggery before experiments.

After the pigs were fed to 32-day, 46-day, and 60-day old, the pigs wereadministrated 2 mL of Bayovac® MH-PRIT-5 (M. hyopneumoniae PRIT-5)vaccine via intramuscular injection. Then, the pigs were continuouslyfed to 74-day old and blood was collected from a jugular vein thereof.The collected blood was placed in room temperature for 1 hour and storedin 4° C. In the next day, the collected blood was centrifugated at1,107×g for 30 minutes and the supernatant was removed to a clean tubeand stored in −20° C.

Two-Dimensional Gel Protein Electrophoresis of the Total Protein ofMycoplasm Spp.

ReadyPrep™ protein extraction kit (total protein) (Bio-Rad, CA, USA) wasused for extracting the total protein of Mycoplasm spp. Afterward, theconcentration of the protein collected was determined by using a Bio-RadRC DC Protein Assay Kit (CA, USA). The detailed protocol can be referredfrom the product description or can be modified from well-knownprotocols in the field.

The two-dimensional gel protein electrophoresis was conducted in twosteps: isoelectric focusing (IEF) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). IEF was toseparate proteins in the sample in view of isoelectric point thereof;SDS-PAGE was to separate proteins accordance with molecular weightthereof. Please see FIG. 1, which shows the result of thetwo-dimensional gel protein electrophoresis.

Hybridization

The serum obtained in step (1) was used as 1^(st) antibody to hybridizewith the result of the two-dimensional gel protein electrophoresis instep (2). After being amplified by 2^(nd) antibody and developed by thefollowing development procedure, proteins showing positive werecollected. Those proteins were recognized by the anti-Mycoplasm spp.antibody and therefore would be suitable as candidate antigens foractive ingredient of subunit vaccines.

The hybridization was conducted by Western blotting. Briefly, the 2D gelafter electrophoresis was transferred to a PVDF membrane. Then, themembrane was incubated and hybridized sequentially with 1^(st) antibody(the serum containing anti-Mycoplasm spp. antibody) and 2^(nd) antibody(AP-conjugated anti-pig IgG). Afterward, a color reaction was conductedby using NBT/BCIP solution.

The result of the color reaction of Western blotting was shown in FIG.2; wherein 10 proteins positive to the immuno-hybridization withanti-Mycoplasm spp. antibody were marked as candidate antigens for beingused as active ingredients of subunit vaccines.

Identification of the Candidate Antigens Obtained

According to the color reaction of the Western blotting, the gelcorresponding to the positive location on the membrane was cut bymicropeptide and analyzed by mass spectrometry. The obtained data of themass spectrometry was then matched with amino acid sequence and proteindatabase to identify those proteins.

Please see the following table 1, said 10 proteins positive to theimmune-hybridization with anti-Mycoplasm spp. antibody were listed.

TABLE 1 the 10 proteins positive to the immune-hybridization withanti-Mycoplasm spp. antibody and amino sequence thereof. Candidate NameSEQ ID NO 1 XylF (xylose-binding lipoprotein) SEQ ID NO: 09 2 XylF(xylose-binding lipoprotein) SEQ ID NO: 09 3 XylF (xylose-bindinglipoprotein) SEQ ID NO: 09 4 PdhA (pyruvate dehydrogenase E1-alpha SEQID NO: 08 subunit) 5 Mhp145 (periplasmic sugar-binding SEQ ID NO: 11protein) 6 EutD (phosphotransacetylase) SEQ ID NO: 10 7 EutD(phosphotransacetylase) SEQ ID NO: 10 8 Mhp389 SEQ ID NO: 14 9 P78(lipoprotein) SEQ ID NO: 12 10 P132 SEQ ID NO: 13 * XylF and EutD havedifferent charge states in cells and therefore become 3 and 2 positivelocation on the membrane.

Example 2 Expressing of Said Candidate Antigens in Large Amount by E.coli Gene Expression System

Escherichia coli JM109 was used as the host cells for cloning andEscherichia coli BL21 (DE3) was used as the host cells for proteinexpression. The Escherichia coli cells were cultured in LB medium(Luria-Bertani; Difco, Mich., USA). According to the experiment design,a proper amount of antibiotic or agar of 1.5% was added to formulating asolid medium.

Amplification of the Genes Encoding the Candidate Antigens

After the candidate antigens were identified, the genes encoding thoseantigens were searched in the NCBI database (National Center forBiotechnology Information). Specific primers targeting the antigen geneswere designed accordingly. Then, the antigen genes were amplified byusing the specific primers and the chromosome of M. hyopneumoniae PRIT-5as template. The specific primers used were listed in the followingtable 2.

TABLE 2  Primer set. Candidate Sequences of the primer set PdhAPdhAF (SEQ ID NO: 15) 5′-GATATAGGATCCATGGACAAATTTCGCTATGTAAAGC CT G-3′PdhAR (SEQ ID NO: 16) 5′-CAATATGTCGACTTATTTTACTCCTTTAAAAAATTCAAGCGCTTC-3′ XylF XylFF (SEQ ID NO: 17)5′-GATATAGGATCCATGAATGGAATAAATTTCTTGGCTT AGGCTTAGTTTTTC-3′XylFR (SEQ ID NO: 18) 5′-CAATATGTCGACTTAATTTTTATTAATATCGGTAATTAGTTTGTCTAAGC-3′ EutD EUTDF (SEQ ID NO: 19)5′-GATATAGGATCCATGACATACCAAGAATATCTTCAAG CAAG-3′) EUTDR (SEQ ID NO: 20)5′-CAATATGTCGACCTATTTACCTTCTTCAAC TTGTAGAGCGCT-3′) Mhp145Mhp145F (SEQ ID NO: 21) 5′-GATATAGGATCCATAGCTTCAAGGTCGAA TACAACTGC-3′Mhp145R (SEQ ID NO: 22) 5′-CAATATGTCGACTTAATTTACCTTTTGGAGTATCCCATTTTC-3′ P78 P78F (SEQ ID NO: 23)5′-GATATAGGATCCTTATCCTATAAATTTAGG CGTTTTTTCC-3′ P78R (SEQ ID NO: 24)5′-CAATATGTCGACTTATTTTGATTTAAAAGCAGGACCT AA AT-3′ P132P132F (SEQ ID NO: 25) 5′-GATATAGGATCCATTGGACTAACAATTTTTGAGAAATCATTTAG-3′ P132R (SEQ ID NO: 26) 5′-CAATATGTCGACTTATTCCTAAATAGCCCCATAAAGTG-3′ Mhp389 Mhp389F (SEQ ID NO: 27)5′-GATATAGGATCCATGGACAAATTTTCACGA ACTGTTCT-3′ Mhp389R (SEQ ID NO: 28)5′-CAATATGTCGACCTAGATTTTAAAGGATTTTTTTAAT TCAATAATATAATC-3′

Polymerase chain reaction (PCR) was conducted with the primer setslisted in the table 2 above to amplify the genes of the candidateantigens. The amplified genes were then used in the E. coli geneexpression system. The PCR condition was: 5 minutes in 98° C. (oneround); 30 seconds in 94° C., 30 seconds in 55° C., X seconds in 68° C.(35 rounds); 5 minutes in 68° C. (one round). Said X was the elongationtime for the DNA polymerase and was set depending on the size of thefragment to be amplified. After the PCR reaction, an electrophoresis wasconducted to verify if the PCR products contained the DNA fragments ofexpected size. Please see FIG. 3, which shows the electrophoresis resultof the PCR products; wherein lane 1 was eutD gene; lane 2 was pdhA; lane3 was xylF; lane 4 was P78 gene; lane 5 was P132 gene; lane 6 wasmhp145; lane 7 was mhp389.

Cloning of the PCR Products

The cloning was conducted by using a CloneJET PCR Cloning Kit, and theligation mixture was transformed into E. coli ECOS™ 9-5 (Yeastern,Taipei, Taiwan). The detailed protocol can be referred from the productdescription or modified from the well-known protocol in the field.

After transformation, the bacteria were cultured on a solid LB mediumcontaining ampicillin (100 μg/mL) until colony thereof formed. Then,colony PCR was conducted to screen strains success in transformation.The PCR condition was: 5 minutes in 95° C. (one round); 30 seconds in95° C., 30 seconds in 55° C., X seconds in 72° C. (25 rounds); 7 minutesin 72° C. (one round). Said X was the elongation time for the DNApolymerase and was set depending on the size of the fragment to beamplified. The elongation speed of Taq DNA polymerase (Genomics, Taipei,Taiwan) is 1 kb/min; therefore, if Taq DNA polymerase is used foramplifying a 1 kb DNA fragment, said X shall be set as 1 minute.

The plasmids of strains, whose recombinant plasmids were verified havingthe insert DNA, were then proceeded to DNA sequencing (Total SolutionProvider of Systems Biology and Chemoinformatics Ltd.). Plasmidscontaining eutD, pdhA, xylF, P78 gene, P132 gene, mhp145, and mhp389were named as pJET-eutD, pJET-pdhA, pJET-xylF, pJET-P78, pJET-P132,pJET-mhp145, pJET-mhp389, respectively.

Point Mutation and Cloning of the Antigen Genes of M. hyopneumoniae

Before amplifying the candidate antigens in an E. coli gene expressionsystem, the codon usage in different organisms shall be considered. Thatsaid, if the gene contains codon that would be encoded ambiguouslybetween the original organism therefrom and E. coli, the gene shall bemodified by point mutation.

The M. hyopneumoniae antigen genes, pdhA, xylF, P78 gene, P132 gene,mhp145, and mhp389, contain TGA codon (eutD does not have the concern incodon usage like others). The TGA codon was translated into tryptophanin Mycoplasma spp. but translated as stop codon in E. coli. In order toprevent from not being able to produce the entire protein in an E. coligene expression system, primers targeting the TGA site were designed andpoint mutation replacing TGA with TGG was conducted by using overlappingextension polymerase chain reaction. As a result, the genes to beexpressed in the E. coli gene expression system can be truthfullytranslated into the candidate antigen of the present invention. Besides,the cutting sites of BamHI of P78 gene, P132 gene, and mhp389 wereundergone silent mutation for the convenience of cloning.

The primers used for point mutation was designed to locate the site ofpoint mutation at the central part of the primer and to have a Tm valueof higher than 78° C. The Tm value of the primers for point mutation wascalculated by using the formula provided by Invitrogene Co.:Tm=81.5+0.41 (% GC)−675/N−% mismatch; wherein % GC is referred as thepercentage of GC in view of the total nucleotides contained in theprimer concerned; N is referred as the length of the primer concerned; %mismatch is referred as the percentage of the base to be mutated in viewof the total nucleotides contained in the primer concerned. The primersets used for the aforesaid genes were listed in the following Table 3to Table 8.

TABLE 3  The primer sets for point mutation of pdhA. PrimerDNA sequence (5′ to 3′) PdhAF GATATAGGATCCATGGACAAATTTCGCTATGTAASEQ ID NO: 29 AGCCTG PdhAM1 GCTAACAAAAGATGACTGGTTTGTCCCAGCTTTTSEQ ID NO: 30 CG PdhAM2 CGAAAAGCTGGGACAAACCAGTCATCTTTTGTTA SEQ ID NO: 31GC PdhAM3 CTTGCAAATGCAATATTGGAATGGTAGCGAAAAA SEQ ID NO: 32 GG PdhAM4CCTTTTTCGCTACCATTCCAATATTGCATTTGCA SEQ ID NO: 33 AG PdhAM5CGAGGCGCTAAATATTGCAAGTATTTGGAAATGG SEQ ID NO: 34CCAGTTGTTTTTTGCGTAAATAAC PdhAM6 GTTATTTACGCAAAAAACAACTGGCCATTTCCAASEQ ID NO: 35 ATACTTGCAATATTTAGCGCCTCG PdhAM7GTTTTTTGCGTAAATAACAATCAATGGGCAATTT SEQ ID NO: 36 CAACCCCAAATAAATATGPdhAM8 CATATTTATTTGGGGTTGAAATTGCCCATTGATT SEQ ID NO: 37GTTATTTACGCAAAAAAC PdhAM9 GTTGAGTTTGTAACTTGGCGTCAAGGTGTTCATASEQ ID NO: 38 CC PdhAM10 GGTATGAACACCTTGACGCCAAGTTACAAACTCASEQ ID NO: 39 AC PdhAM11 GAGAACACGAAAAATGGGAACCAATGCACCGG SEQ ID NO: 40PdhAM12 CCGGTGCATTGGTTCCCATTTTTCGTGTTCTC SEQ ID NO: 41 PdhAM13CCGAAAAACAAAAAATTTGGGATGAAGCGCTTGC SEQ ID NO: 42 GATTG PdhAM14CAATCGCAAGCGCTTCATCCCAAATTTTTTGTTT SEQ ID NO: 43 TTCGG PdhARCAATATGTCGACTTATTTTACTCCTTTAAAAAAT SEQ ID NO: 44 TCAAGCGCTTC

TABLE 4  The primer sets for point mutation of xylF. PrimerDNA sequence (5′ to 3′) XylFF GATATAGGATCCATGAAATGGAATAAATTTCTTGSEQ ID NO: 45 GCTTAGGCTTAGTTTTTC XylFM1CATTTAACCAATCAAGTTGGGAGGCAATTCAACA SEQ ID NO: 46 ACTTGG XylFM2CCAAGTTGTTGAATTGCCTCCCAACTTGATTGGT SEQ ID NO: 47 TAAATG XylFM3CTAATACCAACAAAAATGTTTGGGTACTTTCTGG SEQ ID NO: 48 TTTTCAACACG XylFM4CGTGTTGAAAACCAGAAAGTACCCAAACATTTTT SEQ ID NO: 49 GTTGGTATTAG XylFM5CGGTGATGCGATCACAAAATGGTTAAAAATCCCT SEQ ID NO: 50 GAAAATAAGC XylFM6GCTTATTTTCAGGGATTTTTAACCATTTTGTGAT SEQ ID NO: 51 CGCATCACCG XylFM7TTATCATACTCGGAATTGACTGGACTGATACTGA SEQ ID NO: 52 AAATGTAATTC XylFM8GAATTACATTTTCAGTATCAGTCCAGTCAATTCC SEQ ID NO: 53 GAGTATGATAA XylFM9GAAGAAGCCGGATGGCTTGCAGGATATGC SEQ ID NO: 54 XylFM10GCATATCCTGCAAGCCATCCGGCTTCTTC SEQ ID NO: 55 XylFM11GGTTATCTAGCCGGAATTAAAGCTTGGAATCTAA SEQ ID NO: 56 AAAATTCTGATAAAAAAACXylFM12 GTTTTTTTATCAGAATTTTTTAGATTCCAAGCTT SEQ ID NO: 57TAATTCCGGCTAGATAACC XylFR CAATATGTCGACTTAATTTTTATTAATATCGGTASEQ ID NO: 58 ATTAGTTTGTCTAAGC

TABLE 5  The primer sets for point mutation of P78 gene. PrimerDNA sequence (5′ to 3′) P78F GATATAGGATCCTTATCCTATAAATTTAGGCGTTTTSEQ ID NO: 59 TTCC P78M1 CAATTAATAAAGTTTTGTTTGGTTGGATGATTAATASEQ ID NO: 60 AAGCACTTGCTGATCC P78M2GGATCAGCAAGTGCTTTATTAATCATCCAACCAAAC SEQ ID NO: 61 AAAACTTTATTAATTGP78M3 GATATTAAAGAAATTGAAAGAATCTGGAAAAAATAT SEQ ID NO: 62GTCTCCGATGATCAAGG P78M4 CCTTGATCATCGGAGACATATTTTTTCCAGATTCTTSEQ ID NO: 63 TCAATTTCTTTAATATC P78M5 GCCCTTTCAGGAGGCTCCACTGATTCGGCASEQ ID NO: 64 P78M6 TGCCGAATCAGTGGAGCCTCCTGAAAGGGC SEQ ID NO: 65 P78M7GCCGCAAAAGCTTTTGTTAAATGGCTTTTGACAGAA SEQ ID NO: 66 AAAATAGTCT P78M8AGACTATTTTTTCTGTCAAAAGCCATTTAACAAAAG SEQ ID NO: 67 CTTTTGCGGC P78RCAATATGTCGACTTATTTTGATTTAAAAGCAGGACC SEQ ID NO: 68 TAAAT

TABLE 6  The primer sets for point mutation of P132 gene. PrimerDNA sequence (5′ to 3′) P132F GATATAGGATCCATTGGACTAACAATTTTTGAGAAASEQ ID NO: 69 TCATTTAG P132M1 CTAACTTCTCTAAAAGGTTGGAAAGAAGAAGATGATSEQ ID NO: 70 TTTG P132M2 CAAAATCATCTTCTTCTTTCCAACCTTTTAGAGAAGSEQ ID NO: 71 TTAG P132M3 CTTTCTATTACTTTTGAACTCTGGGACCCAAATGGTSEQ ID NO: 72 AAATTAGTATC P132M4 GATACTAATTTACCATTTGGGTCCCAGAGTTCAAAASEQ ID NO: 73 GTAATAGAAAG P132M5 CCCTGAAGGAGATTGGATAACTTTAGGGAGSEQ ID NO: 74 P132M6 CTCCCTAAAGTTATCCAATCTCCTTCAGGG SEQ ID NO: 75 P132M7CTACCAGGAACTACCTGGGATTTCCATGTTGAAC SEQ ID NO: 76 P132M8GTTCAACATGGAAATCCCAGGTAGTTCCTGGTAG SEQ ID NO: 77 P132M9GGACAACTAATTTGGAGCCAGTTAGCTTCC SEQ ID NO: 78 P132M10GGAAGCTAACTGGCTCCAAATTAGTTGTCC SEQ ID NO: 79 P132M11GGAACAAAAAAGGAATGGATTCTTGTAGGATCTGG SEQ ID NO: 80 P132M12CCAGATCCTACAAGAATCCATTCCTTTTTTGTTCC SEQ ID NO: 81 P132M13CCAATACGCAAATATGGATAACCCGTCTAGGAAC SEQ ID NO: 82 P132M14GTTCCTAGACGGGTTATCCATATTTGCGTATTGG SEQ ID NO: 83 P132M15CCAAGGGGAAGTTCTCTGGACTACTATTAAATCCAA SEQ ID NO: 84 AC P132M16GTTTGGATTTAATAGTAGTCCAGAGAACTTCCCCTT SEQ ID NO: 85 GG P132M17CAAAAAACTTCACCTTTGGTGGATTGCTAATGATAG SEQ ID NO: 86 C P132M18GCTATCATTAGCAATCCACCAAAGGTGAAGTTTTTT SEQ ID NO: 87 G P132RCAATATGTCGACT TATTCCTAAATAGCCCCATAAA SEQ ID NO: 88 GTG

TABLE 7  The primer sets for point mutation of mhp145. PrimerDNA sequence (5′ to 3′) Mhp145F GATATAGG ATCCAT AGCTTCAAGGTCGAATACAASEQ ID NO: 89 CTGC Mhp145M1 AATAATTGCAGAAAAAATTCTTAAAGATCAATGGAASEQ ID NO: 90 AACAAGTAAATATTCTGATTTTTATTCACAAT Mhp145M2ATTGTGAATAAAAATCAGAATATTTACTTGTTTTCC SEQ ID NO: 91ATTGATCTTTAAGAATTTTTTCTGCAATTATT Mhp145RCAATATGTCGACTTA ATTTACCTTTTGGAGTATCC SEQ ID NO: 92 CATTTTC

TABLE 8  The primer sets for point mutation of mhp389. PrimerDNA sequence (5′ to 3′) Mhp389F GATATAGGATCCATGGACAAATTTTCACGAACTGTTSEQ ID NO: 93 CT Mhp389M1 CAATAGTGACAATGGACCCCCCAAATGTTGGTCGSEQ ID NO: 94 Mhp389M2 CGACCAACATTTGGGGGGTCCATTGTCACTATTG SEQ ID NO: 95Mhp389M3 GATAAAGGCGCATCATGGCTTGCGCTTGCACCAAC SEQ ID NO: 96 Mhp389M4GTTGGTGCAAGCGCAAGCCATGATGCGCCTTTATC SEQ ID NO: 97 Mhp389M5GGAAAACTTAAAGGTAAATGGACTTTTGGACTAACC SEQ ID NO: 98 TATTT Mhp389M6AAATAGGTTAGTCCAAAAGTCCATTTACCTTTAAGT SEQ ID NO: 99 TTTCC Mhp389RCAATATGTCGACCTAGATTTTAAAGGATTTTTTTAA SEQ ID NO: 100 TTCAATAATATAATC

The method for the point mutation was briefly explained as follows. Thechromosome of M. hyopneumoniae PRIT-5 was used as template and DNAfragments was amplified by using the primer sets set forth in the table3 to table 8 above.

The 50 μL PCR reaction mixture comprised 1×GDP-HiFi PCR buffer, 200 μMof mixture of dATP, dTTP, dGTP, and dCTP, 1 μM of primers, 100 ng ofchromosome of M. hyopneumoniae PRIT-5, and 1 U of GDP-HiFi DNApolymerase. The PCR condition was: 5 minutes in 98° C. (one round); 30seconds in 94° C., 30 seconds in 55° C., X seconds in 68° C. (35rounds); 5 minutes in 68° C. (one round). Said X was the elongation timefor the DNA polymerase and was set depending on the size of the fragmentto be amplified. The elongation speed of GDP-HIFI DNA polymerase(GeneDirex, Las Vegas, USA) is 1 kb/15 seconds; therefore, if GDP-HIFIDNA polymerase is used for amplifying a 1 kb DNA fragment, said X shallbe set as 15 seconds. After the PCR reaction, an electrophoresis wasconducted to verify if the PCR products contained the DNA fragments ofexpected size. Then, the PCR product was recycled by using a Gel-M™ gelextraction system kit.

Afterward, the PCR product was used as template and amplified by usingthe primer sets set forth in the table 2 above. The PCR condition was: 2minutes in 98° C. (one round); 30 seconds in 94° C., 30 seconds in 55°C., X seconds in 68° C. (35 rounds); 5 minutes in 68° C. (one round).Said X was the elongation time for the DNA polymerase and was setdepending on the size of the fragment to be amplified. The elongationspeed of GDP-HIFI DNA polymerase (GeneDirex, Las Vegas, USA) is 1 kb/15seconds; therefore, if GDP-HIFI DNA polymerase is used for amplifying a1 kb DNA fragment, said X shall be set as 15 seconds. After theaforesaid amplification step, a full length sequence of the candidateantigen genes with point mutation can be obtained.

Then, the PCR product was recycled by using a PCR-M™ Clean Up system kit(GeneMark, Taichung, Taiwan) and the cloning thereof was conducted byusing a CloneJET PCR Cloning Kit. Colony PCR was conducted to confirmthe strains after transformation containing plasmid having the insertDNA and then the plasmids therein were isolated for DNA sequencing(Total Solution Provider of Systems Biology and Chemoinformatics Ltd.).Plasmids containing mutated candidate antigen genes were named aspJET-pdhAM, pJET-xylFM, pJET-P78M, pJET-P132M, pJET-mhp145M,pJET-mhp389M, respectively.

According to the result of sequencing, the DNA sequences of thecandidate antigen genes after point mutation were as shown in SEQ IDNO:01 (pdhA), SEQ ID NO:02 (xylF), SEQ ID NO:03 (eutD, was notpoint-mutated), SEQ ID NO:04 (mhp145), SEQ ID NO:05 (P78 gene), SEQ IDNO:06 (P132 gene), SEQ ID NO:07 (mhp389).

Construction of the Expression Vectors for Expressing the M.hyopneumoniae Antigens

In this part of experiments, plasmid pET-MSY was used as backbone forconstructing an expression vector for expressing M. hyopneumoniaeantigen. pET-MSY is a derivative of pET29a and has a E. coli msyB.Therefore, the expressed recombinant antigen thereby would have a fusionpartner MsyB. MsyB is rich in acidic amino acid and is able ofincreasing the solubility of the protein expressed.

After pJET-eutD, pJET-pdhA, pJET-xylF, pJET-P78, pJET-P132, pJET-mhp145and pJET-mhp389 being digested by BamHI and SalI, DNA fragment obtainedwas inserted into pET-Msy digested previously with the same restrictionenzymes by ligase. Then, the pET-Msy with the DNA fragment wastransformed into E. coli ECOS 9-5. Colony PCR was conducted to confirmthe strains after transformation containing plasmid having the insertDNA and then the plasmids therein were isolated for DNA sequencing(Total Solution Provider of Systems Biology and Chemoinformatics Ltd.).Plasmids verified with correct DNA sequence were named as pET-MSYEutD,pET-MSYPdhA, pET-MSYXylF, pET-MSYP78, pET-MSYP132, pET-MSYMhp145, andpET-MSYMhp389, respectively. Those plasmids obtained were examples ofthe expression vectors for preventing Mycoplasma spp. infection of thepresent invention.

Expression and Isolation of the M. hyopneumoniae Antigens

The vectors for antigen expression were transformed into E. coli BL21(DE3). Single colony of consequent strains after transformation wasinoculated in LB liquid medium containing kanamycin (workingconcentration: 30 μg/mL). After culture overnight at 37° C., 180 rpm,the suspension of the bacteria was diluted at ratio of 1:100 andinoculated again in another LB liquid medium containing kanamycin(working concentration: 30 μg/mL). The bacteria were cultured at 37° C.,180 rpm until OD₆₀₀ therefore achieving about 0.6 to 0.8. Then, 0.1 mMof IPTG was added to induce expression. After induction for 4 hours,pellet was collected by centrifugation (10000×g, 10 minutes, 4° C.) andthe expression was examined via protein electrophoresis.

Afterward, immobilized-metal affinity chromatography (IMAC) was used forprotein isolation through the covalent bonding between the His tag ofthe N-terminal of the recombinant protein and nickel ions or cobaltions. The protocol of protein isolation was in accordance with theproduct description of the QIAexpressionist™ (fourth edition, Qiagen).The pellet was suspended in a lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl,10 mM imidazole, pH 8.0) and disturbed by an ultrasonic processor. Aftercentrifugation (8,000×g, 15 minutes), the supernatant was collected tointroduce into a column of 1 mL Ni-NTA resin. The recombinant antigenswould adhere on said resin. Then, 15 mL wash buffer (50 mM NaH₂PO₄, 300mM NaCl, 20 mM imidazole, pH 8.0) was introduced into the column to washthe resin so that nonspecific proteins adhering thereon can be removed.Lastly, 20 mL elution buffer was added (50 mM NaH₂PO₄, 300 mM NaCl, 250mM imidazole, pH 8.0) to wash off the recombinant antigens on the resin;wherein the imidazole of high concentration can compete the binding siteon the resin with the recombinant proteins and thereby cause therecombinant proteins being washed off. The result of isolation was thenexamined by protein electrophoresis.

The candidate antigens of the present invention collected by isolationcan then be used for the following immune trials to confirm theirability to be used as active ingredient of anti-Mycoplasm spp. subunitvaccines.

Example 3 Swine Immune Challenge Experiments of the Candidate Antigensof the Present Invention

In this example, the candidate antigens of the present invention wereused as active ingredient for preparing subunit vaccines and tested forimmune effects thereof in live swine.

Vaccine Preparation

One isolated recombinant antigen or several isolated recombinantantigens were mixed with alumina gel as an adjuvant to prepare a subunitvaccine or a cocktail subunit vaccine. Every dose of the preparedvaccine was of 2 mL in volume and each kind of antigen contained thereinwas of 100 μg.

The following table 9 listed the samples prepared in this example forimmune challenge experiments.

TABLE 9 Samples of vaccine prepared in Example 3 Sample ActiveIngredient (Antigen) 1 PdhA 2 XylF 3 EutD 4 Mhp145 5 P78 6 P132 7 Mhp3898 PdhA + P78 9 XylF + Mhp145

The swine immune challenge experiments would be conducted by usingBayovac® MH-PRIT-5 (made by using M. hyopneumoniae PRIT-5, as a positivecontrol group), subunit vaccines (samples 1-7 of the present invention),and cocktail vaccines (samples 8 and 9 of the present invention).

33 SPF pigs of 4-week old were brought from Agricultural TechnologyResearch Institute and fed with same feed, environment, and growthcondition in piggery before experiments.

After the pigs were fed to 35-day and 49-day old, the pigs wereadministrated 2 mL of vaccine above via intramuscular injection.

Challenge Experiments

The aforesaid pigs being induced immune response were challenged byMycoplasm spp. at 109-day old to confirm the immune effect of theaforesaid vaccines.

First of all, a lung collected from pigs infected by Mycoplasm spp. wasground in 20 mL of Friis medium and centrifugated at 148.8×g for 10minutes. The supernatant was removed to a clean tube and centrifugatedagain at 7,870×g for 40 minutes. Then, the supernatant was discarded andthe precipitation was suspended in 6 mL of Friis medium to obtain asuspension. Afterward, the suspension was filtered by membrane of 5 μmand 0.45 μm sequentially to obtain bacteria solutions required for thechallenge experiments.

The bacteria solution (5 mL) was administrated to narcotized pigs viatrachea thereof. After 28 days from administration, the pigs weresacrificed and dissected to collect lung thereof. The immune effect wasexamined by observing the lung and recorded according to the followingcriteria: any of meddle upper lobes and upper lobes of any side of thelung observed of pathological trait was scored as 10 points; any ofmeddle upper lobe and diaphragmatic lobes of any side of the lungobserved of pathological trait was scored as 5 points. The full scorewas 55 points. The observation records were shown in FIG. 4.

In comparison with the results of non-injected pigs, the seven candidateantigens of the present invention were able to provide equivalent immuneeffects as conventional vaccine (Bayovac® MH-PRIT-5). If the highersafety of subunit vaccines is taking into consideration, the vaccinescontaining the candidate antigens of the present invention shall bevalued more.

On the other hand, it was not common to use two or more antigens thatwould induce immune effects in one vaccine because the two or moreantigens may not provide doubled immune effect. In fact, there is higherchance that the two or more antigens may interfere or against each otherand consequently reduce the immune effect of the vaccine. According tothe result of this example, sample 8 and sample 9 of the presentinvention (i.e. cocktail vaccine) unexpectedly provide significantincrease in the immune effect. That said, the subunit vaccines of thepresent invention not only have high safety but also provide betterimmune effect when the candidate antigens of the present invention areused in combination.

Those having ordinary skill in the art can readily understand anypossible modifications based on the disclosure of the present inventionwithout apart from the spirit of the present invention. Therefore, theexamples above shall not be used for limiting the present invention butintend to cover any possible modifications under the spirit and scope ofthe present invention according to the claims recited hereinafter.

What is claimed is:
 1. A composition for preventing a disease caused byMycoplasma spp., comprising: an active ingredient, comprising a proteinof Mhp145; and a pharmaceutically acceptable adjuvant; wherein saidMhp145 comprises the sequence of SEQ ID NO:
 11. 2. The composition ofclaim 1, wherein said active ingredient comprises XylF and Mhp145,wherein said XylF comprises the sequence of SEQ ID NO:
 09. 3. Thecomposition of claim 1, wherein said active ingredient is of aconcentration of 50 to 3500 μg/mL based on the total volume of saidcomposition.
 4. The composition of claim 1, wherein saidpharmaceutically acceptable adjuvant is a complete Freund's adjuvant, anincomplete Freund's adjuvant, an alumina gel, a surfactant, a polyanionadjuvant, a peptide, an oil emulsion, or a combination thereof.
 5. Thecomposition of claim 1, further comprising a pharmaceutically acceptableadditive.
 6. The composition of claim 5, wherein said pharmaceuticallyacceptable additive is a solvent, a stabilizer, a diluent, apreservative, an antibacterial agent, an antifungal agent, an isotonicagent, a absorption delaying agent, or a combination thereof.